Parasitology Research (2019) 118:2409–2417 https://doi.org/10.1007/s00436-019-06372-0

PROTOZOOLOGY - ORIGINAL PAPER

In vitro cultivation of Babesia duncani (: ), a zoonotic hemoprotozoan, using infected blood from Syrian (Mesocricetus auratus)

Kimberly A. McCormack1,2 & Amer Alhaboubi3,4 & Dana A. Pollard3,5 & Lee Fuller6 & Patricia J. Holman2

Received: 16 November 2018 /Accepted: 5 June 2019 /Published online: 13 June 2019 # Springer-Verlag GmbH Germany, part of Springer Nature 2019

Abstract Human , a -borne disease similar to , is most often caused by the hemoprotozoans in Europe, and and Babesia duncani in North America. Babesia microti is the best documented and causes more cases of human babesiosis annually than all other agents combined. Although the agents that cause human babesiosis are considered high-risk in transfusion medicine, federally licensed diagnostics are lacking for B. duncani in both the USA and Canada. Thus, there has been a need to develop and validate diagnostics specifically for this . In this study, B. duncani (WA1 isolate) was cultivated in vitro from Syrian (Mesocricetus auratus) infected blood. We hypothesized HL-1 media with supplements would result in B. duncani propagating at higher levels in culture than supplemented M199 similar to the medium the parasite was originally cultivated with in 1994. We were unable to recreate Thomford’s cultivation results with the M199 medium but supplemented HL-1 medium was able to successfully establish continuous culture. We further hypoth- esized that RBC from species other than hamsters would support B. duncani in vitro. However, rat, mouse, horse, and cow RBC did not support continuous culture of the parasite. Culture stocks of B. duncani were deposited at BEI Resources and are now commercially available to the scientific community to further research. The cultured parasite developed in this study was instrumental in the adaptation of B. duncani continuous culture to human RBC.

Keywords Babesia duncani . Human babesiosis . Transfusion . Tick-borne disease . Percentage of parasitized erythrocytes (PPE)

Introduction of human babesiosis and thus are the most clinically relevant (Conrad et al. 2006). B. duncani was initially described in Human babesiosis is a tick-borne disease similar to malaria Washington as WA1 and California as CA5 isolates with an caused by intraerythrocytic protozoan parasites. There are a unknown tick and animal . The search for animal number of recognized zoonotic Babesia spp. in North hosts led researchers to discover mice, gerbils, and hamsters America at present, among which Babesia microti and are susceptible to experimental with B. duncani Babesia duncani (Conrad et al. 2006)causethemostcases (Conrad et al. 2006) (Kjemtrup and Conrad 2000). However,

Section Editor: Leonhard Schnittger

* Kimberly A. McCormack 3 Department of Veterinary Pathobiology, College of Veterinary [email protected] Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843-4467, USA 4 1 Comparative Medicine Program, Department of Veterinary Present address: Department of Parasitology, College of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Medicine, University of Baghdad, Baghdad 10001-10090, Iraq Sciences, Texas A&M University, College Station, TX 77843-4473, 5 Present address: Health Resources and Services Administration, U.S. USA Department of Health and Human Services, 5 Skip Bertman Drive, Baton Rouge, LA 70803, USA 2 Present address: Department of Comparative Medicine, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Boulevard 6 Fuller Laboratories, 1312 East Valencia Drive, Fullerton, CA 92831, Room 203, Oklahoma City, OK 73104, USA USA 2410 Parasitol Res (2019) 118:2409–2417 recent evidence suggests Dermacentor albipictus (Packard, In the current study, blood was collected from highly 1869) (winter tick) and Odocoileus hemionus (Rafinesque, parasitemic hamsters (10–13%). Cultures were subcultured 1817) (mule deer) may fulfill these roles (O’Connor et al. every 2–7 days when blood smears showed elevated PPE 2018;Sweietal.2019). If so, the geographical range of (percentage of parasitized erythrocytes). Our study aimed to B. duncani could be expected to extend to the overlapping compare the growth of B. duncani in HL-1 supplemented regions of this potential tick vector and reservoir host species versus M199 supplemented medium, and to compare the ini- across the USA and Canada. Mule deer are found throughout tiation of cultures directly from blood cryostock versus blood western North America from southern Canada to north of from hamsters with active infection. We further hypothesized central Mexico and as far east as Minnesota and Iowa in the that RBC from species other than hamsters will be able to USA (Innes 2013). The winter tick is found throughout the successfully maintain B. duncani in continuous culture and USA and ranges from its southern-most border in Central mouse, rat, cow, and horse RBC were tested. America to as far north as Alaska (TAMUS 2011). Cases of human babesiosis in the USA have been increas- ing over the last two decades. In 2011, babesiosis was added Materials and methods to the list of nationally notifiable diseases maintained by the Centers for Disease Control and Prevention (CDC) (Herwaldt In vivo et al. 2012). Human babesiosis has been reportable in 27 states since 2013, with 95% of cases occurring in Connecticut, Animals Massachusetts, Minnesota, New Jersey, New York, Rhode Island, and Wisconsin (Moritz et al. 2016). Babesiosis is most Adult male Syrian hamsters (n = 10, > 100 g) (Envigo RMS, commonly acquired from a tick bite. Many infected individ- Inc., Haslet, MI) were pair housed in standard laboratory rat uals are asymptomatic. Those who present with the disease cages (XL Allentown Static Rodent Cage) at Texas A&M have signs ranging from mild flu-like to severe malaria-like University and were fed standard rodent diet (Envigo Teklad symptoms (Herwaldt et al. 2012). It can be appropriately treat- Rodent Diet 8604) and water ad libitum. All housing and ed when correctly diagnosed and usually resolves without procedures were performed in facilities accredited by the complication in immunocompetent patients. The parasite can Association for Assessment and Accreditation of Laboratory easily be transmitted through blood transfusions and cases of Animal Care (AAALAC) and conducted in accordance with transfusion-transmitted babesiosis tend to be more severe or Texas A&M University Institutional Biosafety Permit (IBC even lethal (Lobo et al. 2013). Immunocompromised individ- 2012-133) and approved by the Texas A&M Institutional uals, especially those receiving multiple blood transfusions, Animal Care and Use Committee (IACUC) (AUP 2015- are more at risk for acquiring and developing the severe life- 0256). threatening disease. United States Food and Drug Administration (USFDA) statistics from 2008 to 2012 show Donor blood that babesiosis was responsible for the highest percentage of deaths due to microbial infection resulting from blood trans- Hamster blood for donor RBC was either obtained from indi- fusion (USFDA 2016). The CDC data archives show there vidual hamsters housed at Texas A&M University (TAMU were seven cases of transfusion-associated babesiosis reported hamster) or as a pooled lot from a commercial source in 2012, 14 cases in 2013, and six cases for 2014 (CDC 2015, (BioChemed Services, Winchester, VA). In both cases, blood 2016;Herwaldtetal.2012; Herwaldt and Gray 2016). FDA was collected into EDTA K2. For hamsters housed at Texas statistics show two patients died during 2016 after receiving A&M University, the animals were anesthetized with blood transfusions likely contaminated with Babesia (USFDA isoflurane for retro-orbital venipuncture. A safe amount of 2017). whole blood (0.5–1.7 ml) depending on hamster size was The first report to date of B. duncani (WA1) continuous collectedinto1.3mlK3EDTA microtubes (Sarstedt in vitro culture and the initial characterization was in 1994 #41.1395.105). At least 2 weeks rest was given between ve- (Thomford et al. 1994). Whole infected human blood nipuncture events and collection was from alternate eyes. from a patient was passaged through a Syrian hamster Donor RBC from cow, horse, rat, and mouse were obtained twice. Thomford et al. then initiated cultures in M199 through the Tissue Sharing program at Texas A&M supplemented with FBS using blood from a low University that allows the use of extra blood procured through parasitemic hamster (3.5%). Actively growing parasites other IACUC approved protocols. were subcultured into hamster RBC every 2–3days. All blood was centrifuged at 870×g for3minina Thomford et al. cryopreserved and resuscitated culture- microfuge to pellet the cells, then the plasma and buffy derived piroplasms and showed the pathogenicity was coat were removed and discarded. The cells were washed maintained through hamster inoculation. 3 times by centrifugation in at least 5 volumes of Puck’s Parasitol Res (2019) 118:2409–2417 2411

Saline Glucose (PSG) (Alfa Aesar #J67542) with careful Hamster inoculated to test the infectivity of cultured removal of the supernatant and buffy coat at each wash. B. duncani The washed RBC were stored in an equal volume of PSG plus extra glucose (20 g glucose/L) with a final concen- Cryopreserved passage 25 (P25) B. duncani culture (0.5 ml) tration of 200 μg/ml streptomycin, 200 U/ml penicillin, that had been stored in liquid nitrogen for 34 days was thawed and 50 μg/ml Amphotericin B (Antibiotic/Antimycotic, rapidly in a 37 °C water bath and inoculated i.p. into hamster Atlanta Biologicals #022110) at 4 °C for a maximum of 3. Hamster 3 was monitored daily for the appearance of in- 2weeks. fected RBC as described above.

Hamsters inoculated for culture In vitro

Babesia duncani was procured as cryopreserved WA1 in- B. duncani cultures initiated from BEI cryostock in HL-1 fected hamster blood (NR-12311) from BEI Resources and M199 media (National Institute of Allergy and Infectious Diseases, Manassas, VA). Upon receipt, the vial containing the Cultures were initiated directly from cryopreserved cryopreserved B. duncani was swirled in a 37 °C water B. duncani WA1 infected hamster blood (BEI #NR- bath until the contents were thawed, then the entire con- 12311). The vial containing the frozen infected hamster tents (~ 0.5 ml) were inoculated intraperitoneally (i.p.) in- blood was swirled in a 37 °C water bath until the contents to a Syrian hamster. Day of inoculation was considered were thawed. Immediately thereafter, 225 μlwasdis- day 0. The inoculated hamster was observed daily for any pensed into two culture wells in a 24-well plate (Sigma- clinical signs. The parasitemia was monitored daily by Aldrich #3524) that each contained 70 μlnormalhamster microscopic examination of blood smears stained with donor RBC (BioChemed Services) and 900 μlofmedia. Giemsa at × 1000 under oil immersion. Blood samples The medium in one well consisted of HL-1 medium for the smears were obtained through lateral saphenous (Lonza #77201) supplemented with 20% fetal bovine se- venipuncture and alternating sites were used each day. rum (FBS) (Atlanta Biologicals #S11550), 1 mg/ml At 10 days, when the parasitemia was approximately AlbuMAX I® (Invitrogen #11020–021), 2 mM -gluta- 13%, the hamster was anesthetized with isoflurane and mine (Atlanta Biologicals #B90210), 2% HB101 (v/v, blood collected via terminal cardiac puncture into EDTA reconstituted according to manufacturer’s specifications; and ~ 0.5 ml of the infected blood was inoculated i.p. into Irvine Scientific #T151), 0.2 mM sodium hypoxanthine hamster 2 (Fig. 1) as described in the WA1 information and 0.032 mM thymidine (Hypoxanthine-Thymidine; sheet for in vivo passaging. Part of the collected blood Sigma-Aldrich #H0137-10VL), and a final concentration wasusedtoinitiateB. duncani cultures as below, and part of 200 μg/ml streptomycin, 200 U/ml penicillin, and was cryopreserved as below. The second hamster was 50 μg/ml amphotericin B (Antibiotic/Antimycotic, anesthetized and blood collected via cardiac puncture into Atlanta Biologicals #022110). The second well contained EDTA (Fig. 1) 3 days after inoculation when the medium 199 (M199) with Earle’s balanced salt solution parasitemia was approximately 10%. Part of the blood (Lonza #12-119F) supplemented with 40% FBS, 2 mM - was used for culture as below and part was cryopreserved glutamine, 20 mM TES (2- [Tris(hydroxymethyl)- in 0.5 ml aliquots. methylamino]-ethanesulfonic acid) (TCI America #T0683), and the same antibiotic/antimycotic concentra- tions (Thomford et al. 1994). Each medium was filter sterilized (ThermoScientific Nalgene #165-0045) after all components were added. BEI WA1 For all cultures, the culture medium was replenished daily by removing 800 μl of the spent medium above Hamster 1 Cryopreserve the settled RBC layer and replacing with an equal volume of fresh medium. RBC smears were made daily from each Hamster 2 Culture Hamster 3 culturewell,stainedwithGiemsa,andexaminedat× 1000 under oil immersion microscopy. The percentage of parasitized erythrocytes (PPE) was calculated from a BEI minimum of 500 total erythrocytes. The plates were incu- Fig. 1 Flowchart of B. duncani (WA1) in vivo passages through Syrian bated at 37 °C in a humidified atmosphere of 5% CO2, hamsters (Mesocricetus auratus) and in vitro cultures 2% O2, and 93% N2. 2412 Parasitol Res (2019) 118:2409–2417

B. duncani cultures initiated in HL-1 medium from washed 1 iRBC and 900 μl HL-1 medium. In addition, B. duncani hamster iRBC culture (250 μl) in hamster RBC was subcultured 1:5 into wells containing 70 μl donor washed RBC from cow, horse, Cultures were initiated from B. duncani washed infected RBC rat, or mouse and 900 μlHL-1medium.Thecultureswere (iRBC) within 2 h of collection from hamster 1. Duplicate maintained and monitored as described above. wells received 25 μl of iRBC, 100 μl of normal hamster RBCs (TAMU hamster), and 900 μlofHL-1medium. Cryopreservation Duplicate wells received 60 μliRBC,60μlofnormalhamster RBCs (TAMU hamster), and 900 μl of HL-1 medium. One Cultures of B. duncani were cryopreserved at a PPE of 6 or well received 125 μl of infected RBCs and 900 μlofHL- greater. The selected cultures were resuspended, collected, 1medium. In addition, one well was initiated from hamster 1 and centrifuged at 870×g for 3 min in a microfuge to pellet iRBC stored at 4 °C for 13 days using 70 μlinoculum,70μl the cells, and then the supernatant was removed. Cold PSG+G of normal hamster RBC (TAMU hamster), and 900 μlHL-1 with 20% PVP-40 (Sigma, St. Louis, MO) as the cryoprotec- medium. Cultures were similarly initiated from hamster 2 tant was added in equal volume to the cell pellet. The cells washed iRBC after 10 days refrigeration as follows. Two were gently resuspended and 0.25–0.5 ml was dispensed into wells received 70 μl iRBC, 70 μl of normal hamster RBC each pre-chilled cryovial. The vials were placed in a − 80 °C (TAMU hamster), and 900 μlHL-1medium. ethanol bath overnight and then transferred to liquid nitrogen Subcultures were performed at either a 1:2 or 1:5 split ratio, storage. depending on the PPE and how rapidly the parasites were growing. Wells were subcultured every 2 to 7 days when the Revival from cryopreservation in HL-1 well PPE reached approximately 1.5–2duringthe2ndto7th subculture and 3.5–19 in later subcultures. After the medium Cryovials of cultured B. duncani piroplasms (A1) were re- was replenished, the cells were resuspended by gentle pipet- moved from liquid nitrogen storage and the contents thawed ting. For a 1:2 subculture, 600 μl of the resuspended culture rapidly by swirling the vials in a 37 °C water bath. Cryovials was added to a well containing 600 μl medium and 50 μl were from passages 5, 7, and 12 and had been cryopreserved donor hamster RBC. For a 1:5 subculture, 250 μl of the re- and stored in liquid nitrogen for 53 days, 44 days, and 26 days, suspended culture was added to a well containing 900 μlme- respectively. The thawed contents were dispensed in 250 μl dium and 70 μl donor hamster RBC. aliquots into wells prepared with 900 μl HL-1 medium and 100 μl of washed donor hamster RBC. The plate was incu-

B. duncani cultures initiated in M199 medium from washed bated at 37 °C in a humidified atmosphere of 5% CO2,2%O2, hamster iRBC and 93% N2. The cultures were given fresh medium daily and monitored by Giemsa-stained examination as de- One culture well was initiated from hamster 1 washed iRBC scribed above. stored at 4 °C for less than 48 h, and another was initiated from the same stored iRBC after 13 d at 4 °C. Two culture wells Images were initiated from hamster 2 iRBC within 2 h of collection. Each well received 70 μl iRBC, 70 μl of normal hamster RBC Figure 2 was captured using an Olympus BX41 microscope (TAMU hamster), and 900 μl M199 medium (as described (Olympus America, Inc. Center Valley, PA) equipped with above). SpotSoftware 5.2 Macro-Photography© 2016 SPOT Two additional culture wells were initiated in M199 medi- Imaging (Diagnostic Instruments, Inc. Avon, MA). um from existing B. duncani cultured in HL-1 medium. One well was subcultured from a B. duncani initial culture at a 1:2 split ratio and one well was subcultured from passage 3 Results B. duncani at a 1:5 split ratio. The cultures were maintained and monitored as described above. In vivo

B. duncani cultures initiated in HL-1 medium Hamsters inoculated for culture and non-hamster iRBC After inoculation with the WA1 inoculum (BEI #NR-12311), Culture wells were made using 60 or 100 μlwasheddonor hamster 1 reached a PPE of 13 on day 10 post-inoculation cow RBC and 60 or 25 μl hamster 1 iRBC, respectively, and (p.i.) and blood was collected. Hamster 2 was inoculated with HL-1 medium for a total volume of 1.25 ml. Horse RBC whole blood from hamster 1 and on day 3 p.i., the PPE was 10. (100 μl) were added to culture wells containing 25 μlhamster Results are summarized in Table 1. Parasitol Res (2019) 118:2409–2417 2413

Fig. 2 B. duncani piroplasms from day 1 initial culture using HL-1 medium and hamster RBC. Several parasite forms are shown. (A) Early ring. (B) Early tetrad. (C) Mature ring. (D) Mature tet- rad. , × 1000 oil immersion

Hamster inoculated to test the infectivity of cultured day 16 post-subculture in the new subculture wells or in B. duncani the original wells (data not shown).

Cryopreserved P25 B. duncani culture (cultivated for 4 months Cultures initiated in HL-1 medium from washed B. duncani prior to cryopreservation and then stored in liquid nitrogen for iRBC 34 days) was inoculated i.p. into hamster 3. PPE observed in blood smears obtained from hamster 3 followed the same B. duncani parasitized erythrocytes and many extracellular timeframe as hamster 1 (Table 1) and both were humanely parasites were evident within 24 h in cultures initiated from euthanized at day 10 post-inoculation when PPE exceeded hamster 1 iRBC in HL-1 medium. One culture initiated with 10. The only clinical signs observed in any of the inoculated 25 μl of infected hamster RBC was subcultured on day 2 at a hamsters were lethargy at peak parasitemia at time of terminal 1:5 split ratio. This line (A1) continued to thrive with passages blood collection for hamsters 1 and 3. 2, 3, and 4 of this subcultured line effected on day 8, 15, and 20, respectively. Later passages were done at similar time intervals as shown in Fig. 4. Throughout the propagation of In vitro this line, both the original well and the subcultured well were continuously maintained. Cultured parasites (Fig. 2)appeared B. duncani cultures initiated from BEI cryostock in HL-1 identical in morphology to B. duncani in vivo. Cultures were and M199 media maintained for over 160 days by subculturing an average of every 4.2 days using donor hamster RBC (Fig. 3). Figure 4 The cultures initiated directly from the BEI cryostock of shows the peak parasitemia reached at time of subculture (□). B. duncani-infected hamster blood showed severe hemoly- Over time, the variability of parasite growth decreased and the sisinbothHL-1andM199mediaonday1afterinocula- cultures consistently needed to be subcultured every 3–4days tion. Many extracellular parasites were seen upon exami- during the last 20 of the 160 days shown in Fig. 4 by the nation of the daily RBC smears. A few rare (0–4 iRBC/500 diamond marker ♦. Cultured B. duncani (A1) was deposited RBC) early ring forms as observed in Abraham et al. with BEI Resources and is available under NR-50440 Babesia (2018) were seen each day until day 14. An average of 2 duncani, WA1 (in vitro) (parasitic ). infected blood cells out of 1000 were seen on day 16 post- B. duncani cultures initiated with 60 or 125 μl infected inoculation. The wells were subcultured 1:5 into single hamster 1 RBC initially propagated similarly as above but new wells for each medium. No parasites were seen after were subcultured only once at days 5 and 2, respectively,

Table 1 Babesia duncani initial and final PPE from passaging Inoculated Inoculum Inoculum Day parasites first Day blood PPE at through Syrian hamsters hamster PPE seen collected collection

1 10% Cryopreserved whole 61013% blood 2 13% Hamster 1 whole 1 3 10% blood 3 12% Cryopreserved P25 51015% culture 2414 Parasitol Res (2019) 118:2409–2417

Fig. 3 B. duncani growth in continuous culture over 160 days. Cultures were initiated from hamster 1 iRBC and maintained in donor hamster RBC and HL-1 medium. On day 66 an exceptionally high of 28 was recorded and is indicated by an asterisk (*). PPE at subcultures are indicated (□)

and then discontinued due to limited supply of hamster donor subpassage, were used as inocula. Results for cow, horse, and RBC (data not shown). rat are shown in Fig. 6. Mouse RBC cultures underwent se- Two cultures initiated with 25 μl of infected hamster 2 vere and excessive cellular debris obscured RBC RBC in HL-1 medium were subcultured on days 2 and 4, on microscopy to the point that PPE could not be calculated. respectively, when the PPE reached approximately 3 None of the alternate donor RBC was able to support in vitro (Fig. 5). All wells were continuously maintained as described cultivation of B. duncani. It was observed that after infected above. One culture became established whereas the other died hamster RBC were introduced into culture with noticeably out after 1 week as shown in Fig. 5. larger RBC, such as horse or cow, the parasites appeared to Medium 199 did not continuously support B. duncani infect residual hamster RBC instead of invading different host in vitro when initiated from either infected hamster 1 or ham- RBC. ster 2 blood, irrespective of whether fresh or stored inoculum was used. Two culture wells from hamster 2 in M199 underwent one subculture but PPE decreased thereafter until Revival from cryopreservation in HL-1 no viable parasites were visible on Giemsa-stained slides. Overall, parasites persisted in some M199 cultures for up to Cryopreserved culture from B. duncani A1 passages 5, 7, and 2 weeks but none of the cultures became established (data not 12 were successfully revived in HL-1 medium with hamster shown). RBC. Free-floating parasites were visible the day after inocu- lation. Intracellular parasites became visible as early ring B. duncani cultures initiated in HL-1 medium forms (Abraham et al. 2018) on stained smears relative to and non-hamster iRBC the passage level at which they were cryopreserved. Early ring forms began to be seen in the P12 revival at day 8 post-inoc- B. duncani in hamster RBC, either blood derived or culture ulation, P7 revival had a few at day 10, and P5 revival showed subpassage, were used as inocula in cultures that used normal visible early ring forms at day 16. All revived cultures were donor RBC from cow, horse, rat, and mouse donor RBC. maintained successfully for over 2 months (data not shown) at B. duncani in hamster RBC, either blood derived or culture which point they were discontinued.

20% 10

9 

deziti 8 15% 7 s

araP%(EPP 6 10% 5 4 3 5% Erythrocytes) □ Erythrocytes) 2 1 0% 0 1 3 5 7 9 111315171921232527293133 subculture last since Days Passages over 160 days

Fig. 4 B. duncani PPE at time of subculture over 35 passages and days parasite growth decreased and the cultures consistently needed between subcultures. Cultures were initiated from infected hamster 2 subcultured every 3–4 days during the last 20 of the 160 days shown in blood and maintained in donor hamster RBC and HL-1or M199 Fig. 4 by the diamond marker (♦) medium. PPE at subcultures are indicated (□). Over time, variability of Parasitol Res (2019) 118:2409–2417 2415

Fig. 5 B. duncani cultured in 20 donor RBC from horse, cow, or )setycorhtyrEde rat using HL-1 medium. PPE on the day of subculture is indicated 15 (□)

10 z itisaraP%(EPP

5

0 12345678910111213 Days Cow Horse Rat

Discussion daily. This rapid increase in parasitemia may resemble the higher parasitemia seen in immunocompromised patients B. duncani continuous cultures were successfully established and other cases of severe disease from B. duncani. when initiated using inoculum from infected Syrian hamsters. One of the challenges to overcome when cultivating B. duncani remained viable for culture in washed erythrocytes Babesia spp. continuous cultures is selecting an appropriate from infected blood for at least 13 days when stored under donor animal for routine blood supply (Alhaboubi et al. 2017). refrigeration. Although B. duncani did not adapt to culture Even within the appropriate species, erythrocytes and/or se- directly from infected hamster blood cryostock, the cryopre- rum from some animals may not be suitable for supporting the served cultured parasites were successfully recovered both parasite in vitro and may inhibit parasite growth (Schuster in vitro and in vivo from liquid nitrogen storage. Parasites 2002; Alhaboubi et al. 2017). It is possible immune factors cryopreserved in the 25th culture passage were infective such as antibodies or cytokines contribute to this difficulty in vivo, with the hamster developing parasitemia similar to even when red blood cells are washed and the buffy coat the course seen with the hamster inoculated with the BEI removed as thoroughly as feasible. B. duncani-infected hamster blood cryostock (BEI #NR- The Syrian hamster is the most frequently used animal 12311). Certain Babesia spp. cultured over time shows de- model for human babesiosis, including B. microti and creased infectivity and as a result, some attenuated strains B. duncani (Souza et al. 2016). Hamster blood obtained from have been adapted as (Schuster 2002). While our commercial sources is usually a pooled sample from multiple current results showed no difference between the hamsters animals. To facilitate repeated blood collection from the same that received 25th passage cultured parasites versus the BEI animals that supported the parasite, a small in-house colony of infected blood cryostock, it remains to be determined whether hamsters was maintained for blood donors. However, only a attenuation will occur as B. duncani undergoes more passages small quantity of hamster blood was retrieved per draw (ap- in vitro. proximately 1 ml whole blood) in order to stay within humane The hamsters inoculated with B. duncani in this study, re- limits. This impacted the number of cultures we could initiate gardless of source, showed a rapid increase in parasitemia and maintain. The need for repeated and larger blood collec- from approximately 2 to 10% or greater within 24 h. The tions encouraged our attempt to experiment with donor blood hamsters were monitored for parasitemia early in the morning more readily available through tissue sharing, which included

Fig. 6 B. duncani cryostocks from P5, P7, and P12 were )setycor revived. PPE on the day of 25 subculture is indicated (□) h

tyrEdezitisaraP% 20

15

10

5 (EP 0

P 1234567891011121314 Days P5 cryostock P7 cryostock P12 cryostock 2416 Parasitol Res (2019) 118:2409–2417 horse, cow, rat, and mouse. However, hamster RBC were the development of more sensitive molecular assays for Babesia only donor RBC that supported parasite propagation and re- spp. in states where babesiosis is endemic (Karkoska et al. sulted in the establishment of B. duncani continuous cultures. 2017). Neither the USA nor Canada has in place federally In the current study, Medium 199 as previously described licensed tests for screening donated blood. Federally licensed for B. duncani in vitro culture (Thomford et al. 1994)was diagnostics are still needed in the USA and Canada for compared with HL-1 medium. In our hands, M199 did not B. duncani blood donor testing and accurate patient diagnosis. support the parasite despite the reported earlier success with The ability to culture Babesia spp. can speed this process by PPEconsistentlypeakingat20to30every2to3days providing a continuous supply of sufficient pathogen to test. (Thomford et al. 1994). The reason for this disparity is un- Continuous cultures of B. duncani were successfully known but possibly may be due more to the fetal bovine established in the current study and were shown to remain serum or the donor hamster RBC used in the different studies viable and infective after cryopreservation. B. duncani cul- rather than to the medium composition. The M199- tures were deposited with BEI Resources and cultured supplemented medium is composed of 40% fetal bovine se- B. duncani is now offered as a resource to other researchers. rum whereas HL-1, which is formulated as an enriched serum- Cultured B. duncani provides a continuous laboratory re- free medium for hybridoma culture, is supplemented with source available for molecular and biochemical studies, anti- only 20%. The enriched formulation of HL-1with additional babesial drug testing, diagnostic tests, and develop- supplementation seems to have provided the required nutri- ment (Canning and Winger 1987;Schuster2002;Alhaboubi ents to allow the establishment of continuous B. duncani cul- et al. 2017; Abraham et al. 2018). It also reduces the overall tures in this study. Another more likely theory is that the high use of animals in research by providing an alternative to main- parasitemia collected from infected hamsters led to a slower taining a source of parasites by passaging through the animal initial growth rate as observed by Abraham et al. (2018). The host. As a result of this study, now one of the causes of human growth curve did become more consistent and increase PPE babesiosis, B. duncani, is available to the scientific communi- over time (Figs. 3 and 4); however, it did not reach the con- ty as a cryostock that can be directly cultured in vitro. The line sistent 20 to 30% PPE every 2 to 3 days as Thomford et al. described herein was utilized by Abraham et al. (2018)to (1994) initially experienced. adapt B. duncani to continuous culture in human RBC for Although lower parasitemias resulted in the current cul- their research into effective clinical therapies. tures, the cultured parasites were robust and the stocks sub- mitted to BEI were expanded to provide a source of cultured Compliance with ethical standards B. duncani to the scientific community. The utility of our cultured line is demonstrated in the work of Abraham et al. Ethical approval All applicable international, national, and/or institu- who tested therapeutic anti-babesial drug efficacy in vitro tional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance (2018). They tested four currently recommended treatments with the ethical standards of Texas A&M University at which the studies for human babesiosis (, azithromycin, were conducted. , and ), and the results suggest these ther- apies have limited efficacy in cases of human babesiosis Conflict of interest The authors declare that they have no conflict of caused by B. duncani infection. B. duncani cultures in hamster interest. RBC established in this current study were utilized for the establishment of the continuous culture in human RBC. As such, the hamster RBC growth medium (HaRGM) is nearly References identical to the supplemented HL-1 medium used in this study except the addition of 100 μg of gentamicin. Other species of Alhaboubi A, He L, McCormack K, Gustafson A, Holman PJ (2017) Recovery of bovine Babesia spp. after long-term cryostorage and Babesia have also been cultivated using a similar medium comparison of bovine donor erythrocytes and serum. 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